Methods and means of histone methylation

ABSTRACT

This invention relates to the methylation of histones, in particular to a previously uncharacterised group of histone H3 methylases which comprise a SET domain and which methylate either lysine 4 within the amino tail of histone H3 or within the histone H3 core. Methylation by these methylases, in particular trimethylation, is shown to be important for transcriptional activity.

This application is the U.S. national phase of international applicationPCT/GB02/02050 filed 2 May 2002, which designated the U.S.PCT/GB02/02050 claims priority to GB Application No. 0111218.4 filed 8May 2001. The entire contents of these applications are incorporatedherein by reference.

This invention relates to histone methylation, in particular, to themethylation of specific lysine residues on histone H3 and to thebiological effects of such methylation.

DNA in the eukaryotic nucleus is wrapped around a histone-core which isa protein complex involving the four histones H4, H3, H2B and H2A. ThisDNA-histone structure (nucleosome) is not compatible with geneexpression. Re-organisation of the nucleosome is required fortranscription factors and RNA polymerase to have access to the DNA fortranscription.

Covalent post-translational modifications of the amino-terminal tails ofhistones regulate the transcriptional ‘on’ or ‘off’ states of chromatinand influence chromosome condensation and segregation.

Such modifications include acetylation, phosphorylation and methylation.

Suv39H1 is a methyltransferase which is specific for lysine 9 of histoneH3 (Rea et al (2000) Nature 406 593–598). Methylation of lysine 9 bySuv39H1 leads to the recruitment of the HP1 repressor protein (Bannisteret al (2001) Nature 410 120–124, Lachner et al (2001) 410 116–120) andthe formation of transcriptionally silent heterochromatin.

Suv39H1 has homologues in human (Suv39H2), Drosophila (su(var)3–9) andS. pombe (clr4). Defects in Suv39H1 and its homologues have beencorrelated with defects in transcription and aberrant mitotic divisionand chromosomal mis-segregation.

Expression of the Suv39h1 protein has also been shown to inhibit cellproliferation of mammalian cells and its phosphorylation status is knownto change as cells progress from G1 to S.

The Suv39h1 methyltransferase comprises an evolutionarily conserveddomain called a SET domain (SUV39 Enhancer of Zest and Trithorax).However, SET domains are also found in other proteins which do not havehistone methylase activity (Rea et al. (2000)).

A histone methylase activity has also been identified in yeast Clr4 thisactivity has been shown to be essential for transcriptional repression(Bannister et al (2001) Nature 410 120–124). Lack of Clr4 methylaseactivity activates heterochromatically repressed genes at centromeresand may lead to chromosome mis-segregation.

The present inventors have discovered that a group of previouslyuncharacterised SET domain containing proteins are histone H3methylases. This group includes S. cerevisiae SET1 (YHR119W) and itshuman homologues hSET1.1P (KIAA0339) and hSET1.2P (KIAA0339) and S.cerevisiae SET2 (YJL168C) and its human homologues hSET2 (AJ238403) andWHSC1 (XP_(—)003388).

The present inventors have further discovered that these methylases haveactivities which are distinct from Suv39h1 and other previouslycharacterised histone methylases. SET1 and related proteins specificallymethylate lysine 4 of histone H3 while SET2 and related proteinsspecifically methylate a lysine residue outside the amino tail region ofhistone H3 (i.e. a core lysne residue).

Various aspects of the present invention provide for the use of a SETpolypeptide and a histone H3 polypeptide in screening methods and assaysfor agents which modulate methylation of histone H3 by the SETpolypeptide, and which may therefore be useful in the modulatingcellular proliferation, for example in treating conditions such ascancer.

In a general aspect, the present invention provides an assay method foran agent with ability to modulate, e.g. disrupt, interfere with, orincrease interaction and/or binding of histone H3 with a SETpolypeptide, the method including:

-   (a) bringing into contact a SET polypeptide and a histone H3    polypeptide; and,-   (b) determining binding and/or interaction between the histone H3    polypeptide and the SET polypeptide.

An assay may be carried out in the presence of a test compound underconditions in which, in the absence of the test compound, the SETpolypeptide will interact or bind with histone H3.

The precise format of the assay of the invention may be varied by thoseof skill in the art using routine skill and knowledge. For example, theinteraction between the polypeptides may be studied in vitro bylabelling one with a detectable label and bringing it into contact withthe other which has been immobilised on a solid support.

Suitable detectable labels include ³⁵S-methionine which may beincorporated into recombinantly produced peptides and polypeptides.Recombinantly produced peptides and polypeptides may also be expressedas a fusion protein containing an epitope which can be labelled with anantibody.

Fusion proteins may be generated that incorporate six histidine residuesat either the N-terminus or C-terminus of the recombinant protein. Sucha histidine tag may be used for purification of the protein by usingcommercially available columns which contain a metal ion, either nickelor cobalt (Clontech, Palo Alto, Calif., USA). These tags also serve fordetecting the protein using commercially available monoclonal antibodiesdirected against the six histidine residues (Clontech, Palo Alto,Calif., USA).

A protein may be immobilized on a solid support using an antibodyagainst that protein bound to a solid support or via other technologieswhich are known per se. A preferred in vitro interaction may utilise afusion protein including glutathione-S-transferase (GST). This may beimmobilized on glutathione agarose beads. In an in vitro assay format ofthe type described above, a test compound can be assayed by determiningits ability to diminish the amount of labelled peptide or polypeptidewhich binds to the immobilized GST-fusion polypeptide. This may bedetermined by fractionating the glutathione-agarose beads bySDS-polyacrylamide gel electrophoresis. Alternatively, the beads may berinsed to remove unbound protein and the amount of protein which hasbound can be determined by counting the amount of label present in, forexample, a suitable scintillation counter.

An assay according to the present invention may also take the form of anin vivo assay. The in vivo assay may be performed in a cell line such asa yeast strain in which the relevant polypeptides or peptides areexpressed from one or more vectors introduced into the cell.

Various methods and uses of modulators which inhibit, potentiate,increase or stimulate methylation of histone H3 by a SET polypeptide areprovided as further aspects of the present invention.

The purpose of disruption, interference with or modulation of themethylation of histone H3 by a SET polypeptide may be to modulatecellular functions such as transcription and proliferation which aremediated by virtue of such methylation, such as transcription, asdiscussed above and further below.

A method of screening for a substance which modulates activity of a SETpolypeptide may include contacting one or more test substances or agentswith the SET polypeptide in a suitable reaction medium, testing theactivity of the treated polypeptide and comparing that activity with theactivity of the polypeptide in comparable reaction medium untreated withthe test substance, substances or agent. A difference in activitybetween the treated and untreated SET polypeptides is indicative of amodulating effect of the relevant test substance or substances.

Thus another aspect of the present invention provides an assay methodfor an agent with ability to modulate, e.g. disrupt, interfere with,increase or stimulate the histone H3 methylase activity of a SETpolypeptide, the method including:

-   (a) bringing into contact a SET polypeptide and a test compound;    and,-   (b) determining the histone H3 methylase activity of the SET    polypeptide.

An assay may be carried out under conditions in which, in the absence ofthe test compound, the SET polypeptide will possess histone H3 methylaseactivity. Histone H3 methylase activity may be determined as describedherein.

The SET polypeptide and the test compound may, for example, be broughtinto contact in the presence of a histone H3 polypeptide. Histone H3methylase activity may then be determined by determining the presence ofone or more methyl groups at a lysine residue of the histone H3polypeptide.

In another aspect, the present invention provides a method foridentifying and/or obtaining an agent with ability to modulate, e.g.disrupt, inhibit, interfere with, increase or stimulate methylation ofhistone H3 by a SET polypeptide, the method including:

-   (a) bringing into contact a SET polypeptide and a histone H3    polypeptide in the presence of a test compound; and,-   (b) determining methylation of the histone H3 polypeptide.

An assay may be carried out under conditions in which, in the absence ofthe test compound, the SET polypeptide will methylate histone H3.

Determining methylation of the histone H3 polypeptide may comprisedetermining the presence or absence of one or more methyl groups on alysine residue of said histone H3 polypeptide, for example lysine 4 or acore lysine residue.

Where the SET polypeptide is a SET1 polypeptide, methylation of thelysine 4 residue of histone H3 may be determined. Where the SETpolypeptide is a SET2 polypeptide, methylation of a lysine residueselected from the group consisting of lysine 27, lysine 36, lysine 37,lysine 42, lysine 56, lysine 64, lysine 79, lysine 115, lysine 121,lysine 122 and lysine 125 within the core of histone H3 may bedetermined. More preferably, where the SET polypeptide is a SET2polypeptide, methylation of lysine 36 may be determined.

A test compound which increases, potentiates, stimulates, disrupts,reduces, interferes with or wholly or partially abolishes methylation ofthe histone H3 polypeptide and which may thereby modulate SETpolypeptide activity, may thus be identified and/or obtained.

Methylation may be determined according to known methods describedherein.

Agents which increase or potentiate methylation of histone H3 may beidentified using conditions which, in the absence of apositively-testing agent, prevent methylation. Such agents may be usedto potentiate the function of a SET polypeptide and may have an effect,for example, on transcription and/or DNA replication.

In methods of the present invention, a histone H3 polypeptide maybrought into contact with a SET polypeptide in the presence of asuitable substrate such as SAM (S-adenosyl-(methyl)-L-methionine). Thesubstrate may be labelled, e.g. (S-adenosyl-(methyl-¹⁴C)-L-methionine)and the amount of label on the histone H3 after incubation with SETpolypeptide determined.

Methods described herein may be useful in determining the presence of,and optionally quantifying, the amount of SET polypeptide in a testsample. This may have a diagnostic or prognostic purpose, e.g. in thediagnosis or prognosis of any medical condition discussed herein (e.g. aproliferative disorder such as cancer) or in the evaluation of a therapyto treat such a condition.

The characterisation of the histone H3 methylase activity of a SETpolypeptide as described herein and its role in the regulation ofcellular proliferation and transcriptional activity allows the use ofmaterials and methods, such as are disclosed and discussed above, forestablishing the presence or absence in a test sample, for example,obtained from an individual, of aberrant, i.e. increased, reduced orabolished SET polypeptide histone H3 methylase activity. Such aberrantactivity may be determined as described herein for the purpose ofdiagnosing a predisposition of an individual to a condition associatedwith cellular proliferation or for diagnosing an individual as sufferingfrom a condition associated with cellular proliferation, such as cancer.The presence of aberrant SET polypeptide histone H3 methylase activitybeing indicative of the individual having a condition associated withcellular proliferation or being predisposed i.e. having an increasedsusceptibility to a condition associated with cellular proliferation.

Aberrant expression may be detected at the protein level, by determiningthe histone H3 methylase activity of a SET polypeptide, as describedherein, for example, the presence or absence or amount of methylaseactivity or at the nucleic acid level (i.e. DNA or RNA), by determiningthe presence of a mutant, variant or allele of a SET gene which encodesa SET polypeptide which has aberrant activity or which expressesaberrant i.e. abolished, reduced or increased levels of SET polypeptide.The presence or amount of SET polypeptide expression may be determinedby determining the presence and/or amount of mRNA encoding thepolypeptide.

In assay methods according to the invention, the amount of testsubstance or compound which may be added to an assay of the inventionwill normally be determined by trial and error depending upon the typeof compound used. Typically, from about 0.001 nM to 1 mM or moreconcentrations of putative inhibitor compound may be used, for examplefrom 0.01 nM to 100 μM, e.g. 0.1 to 50 μM, such as about 10 μM. Greaterconcentrations may be used when a peptide is the test substance. Even amolecule which has a weak effect may be a useful lead compound forfurther investigation and development.

A test substance, compound or agent used in an assay method as describedherein may be comprised in a sample, mixture or extract, for example, abiological sample.

A screening or assay method may include purifying and/or isolating atest substance and/or substance of interest from a mixture or extract,i.e. reducing the content of at least one component of the mixture orextract, e.g. a component with which the test substance is naturallyassociated. The screening or assay method may include determining theability of one or more fractions of a test mixture or extract tomodulate the methylase activity of the SET polypeptide.

The purification and/or isolation may employ any method known to thoseskilled in the art.

The precise format of any of the screening or assay methods of thepresent invention may be varied by those of skill in the art usingroutine skill and knowledge. The skilled person is well aware of theneed to employ appropriate control experiments.

Compounds which may be screened using the assay methods described hereinmay be natural or synthetic chemical compounds used in drug screeningprogrammes. Extracts of plants, microbes or other organisms, whichcontain several characterised or uncharacterised components may also beused.

Combinatorial library technology provides an efficient way of testing apotentially vast number of different substances for ability to modulatean interaction. Such libraries and their use are known in the art, forall manner of natural products, small molecules and peptides, amongothers. The use of peptide libraries may be preferred in certaincircumstances.

Methods of determining the methylation of histone H3 by a SETpolypeptide and of screening for an agent able to modulate themethylation of histone H3 by a SET polypeptide, include methods in whicha suitable end-point is used to assess interaction.

Where the SET polypeptide is a SET1 polypeptide, suitable end pointsinclude the determination of the methylation of the lysine 4 residue ofhistone H3 using methods as described herein.

Where the SET polypeptide is a SET2 polypeptide, suitable end pointsinclude the determination of the methylation of histone H3 outside the Nterminal 26 amino acids (i.e. at a core lysine residue) using methods asdescribed herein, for example methylation of lysine 36.

Methylation may be determined by any convenient method known to askilled person. For example, histone H3 polypeptide or variant orderivative thereof, may be immobilised e.g. on a bead or plate, andmethylation of the appropriate residue detected using an antibody orother binding molecule which binds the N terminal region of histone H3with a different affinity when the residue is methylated from whenresidue is not methylated. Antibodies may also be used to determine thelevel of methylation e.g. di- or tri-methylation, of a histone H3 lysineresidue. Antibodies may be obtained by means of any standard techniqueas discussed elsewhere herein, e.g. using a methylated peptide (such asan N terminal fragment of histone H3 or a fragment with the N terminalamino acids deleted).

Binding of a molecule which discriminates between the methylated andnon-methylated form of a histone H3 polypeptide or between differentdegrees of methylation may be assessed using any technique available tothose skilled in the art, which may involve determination of thepresence of a suitable label.

Methylation may also be assayed in solution, e.g. as described in Rea etal (2000), Nature, 406: 593–599. Briefly, 10 μg of free histonesubstrate (mixture of H1, H2, H3, and H4; Boehringer Mannheim) is mixedwith 300 nCi S-adenosyl-[methyl-¹⁴C]-L-methionine (25 μCi ml⁻¹)(Amersham) as methyl donor in methylase activity buffer (50 mM Tris.HClpH8.5, 20 mM KCl, 10 mM MgCl₂, 10 mM β-mercaptoethanol, 250 mM sucrose),to give a final volume of 50 μl. 10 μg of SET polypeptide preparation isadded and the reaction incubated at 37° C. for 60 mins. The reactionproducts are then resolved by SDS-PAGE and viewed following fluorographyof the gel. Alternatively, following SDS-PAGE, the resolved proteins canbe Western blotted to a nitrocellulose membrane, which is then dried andexposed to film.

Methylation may also be determined as described below in the examples.

Of course, the person skilled in the art will design any appropriatecontrol experiments with which to compare results obtained in testassays.

A histone H3 polypeptide may be a full-length histone H3 protein from aeukaryotic cell, such as a yeast or a mammal, for example a human. Theterm also includes fragments of the full-length protein sequence, suchas fragments which comprise the lysine 4 residue, for example, fragmentscomprising the N terminal residues of the full length protein. Otherfragments may comprise residues from the core of histone H3, i.e.outside the amino tail region, such as fragments which comprise a lysineresidue selected from the group consisting of lysine 27, lysine 36,lysine 37, lysine 42, lysine 56, lysine 64, lysine 79, lysine 115,lysine 121, lysine 122 and lysine 125, for example, a fragmentcomprising lysine 36.

A SET polypeptide may be a full-length SET protein or a fragment thereofwhich retains the methylase activity of the full length SET protein, forexample a polypeptide which comprises the SET domain.

Examples of SET domains are shown in FIGS. 1 and 2. A SET domain maycomprise three conserved motifs; NHSC, GEXSY and Erich (Rhea S. et al(2000) Nature 406 593–599). These motifs are indicated in FIG. 1 andFIG. 2.

A SET domain of a SET1 polypeptide may further comprise conserved motifsI, II, III, IV and V as indicated in FIG. 1 and FIG. 2.

A SET polypeptide may be a member of the SET1 or the SET2 families ofhistone H3 methylases (HMTs).

SET1 polypeptides specifically methylate the lysine 4 residue of histoneH3 and include S. cerevisiae SET1 (Database Accession Number YHR119W),S. pombe SET1 (SPCC306.04c), hSET-1P (Human: KIAA0339), hSET-2P (Human:KIAA1076), HRX, TRX2, KIAA1090, and ALR (all on public databases). SET1polypeptides may comprise a SET domain having greater than 35%, greaterthan 40%, greater than 45% or greater than 50% sequence identity withthe SET domain of S. cerevisiae SET1.

An aspect of present invention provides a SET1 polypeptide whichmethylates lysine 4 of a histone H3 polypeptide.

SET2 polypeptides specifically methylate a lysine residue selected fromthe group consisting of lysine 27, lysine 36, lysine 37, lysine 42,lysine 56, lysine 64, lysine 79, lysine 115, lysine 121, lysine 122 andlysine 125 i.e. a core lysine of histone H3 outside the N terminal 26amino acid tail. More preferably, a SET2 polypeptide specificallymethylates lysine 36. SET2 polypeptides include S. cerevisiae SET2(YJL168C), S. pombe SET2 (SPAC29B12), hSET2 (AJ238403), WHSC1(XP_(—)003388), Q9H6H8, NSD1, NSD3 and ASH1 (all on public databases).

Another aspect of present invention provides a SET2 polypeptide whichmethylates a core lysine residue of a histone H3 polypeptide.

SET2 polypeptides may comprise a SET domain having greater than 35%,greater than 40%, greater than 45% or greater than 50% sequence identitywith the SET domain of S. cerevisiae SET2.

A histone H3 or SET polypeptide may include any suitable fragment,variant, derivative, allele or homologue of histone H3 or the SETpolypeptide, which may be employed in a method described herein.Suitable fragments, variants or derivatives of histone H3 retain thebiological activity of being methylated by a SET polypeptide i.e. theyinclude the appropriate sites of methylation by the SET polypeptide, forexample, lysine 4 or other target residues within the histone H3 core(outside the amino tail residues 1 to 26), for example lysine 36.Suitable variants or derivatives of a SET polypeptide retain the histoneH3 methylase activity.

A polypeptide which is an amino acid sequence variant, allele,derivative or mutant of an amino acid sequence described herein maycomprise an amino acid sequence which shares greater than about 60%sequence identity with the sequence shown, greater than about 70%,greater than about 80%, greater than about 90% or greater than about95%. The sequence may share greater than about 70% similarity, greaterthan about 80% similarity, greater than about 90% similarity or greaterthan about 95% similarity with the amino acid sequence described herein.

For amino acid “homology”, this may be understood to be similarity(according to the established principles of amino acid similarity, e.g.as determined using the algorithm GAP (as described below) or identity.

Amino acid similarity is generally defined with reference to thealgorithm GAP (Accelerys, formerly Genetics Computer Group, Madison,Wis.). GAP uses the Needleman and Wunsch algorithm to align two completesequences that maximizes the number of matches and minimizes the numberof gaps. Generally, the default parameters are used, with a gap creationpenalty=12 and gap extension penalty=4. Use of GAP may be preferred butother algorithms may be used, e.g. BLAST (which uses the method ofAltschul et al. (1990) J. Mol. Biol. 215: 405–410), FASTA (which usesthe method of Pearson and Lipman (1988) PNAS USA 85: 2444–2448), or theSmith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147:195–197), generally employing default parameters.

Similarity allows for “conservative variation”, i.e. substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine. Particular amino acid sequence variants may differ from asequence described herein by insertion, addition, substitution ordeletion of 1 amino acid, 2, 3, 4, 5–10, 10–20 20–30, 30–50, 50–100,100–150, or more than 150 amino acids.

Sequence comparison may be made over the full-length of the relevantsequence shown herein, or may more preferably be over a contiguoussequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133,167, 200, 233, 267, 300, 333, 400 or more amino acids, compared with therelevant amino acid sequence.

As described above, peptides which include or consist of fragments of afull-length SET protein are encompassed within the term ‘SETpolypeptide’ as defined herein. Where the SET polypeptide is a fragmentof the full-length protein, suitable fragments are generally those whichretain the methylase activity of the full-length polypeptide and arecapable of methylating histone H3, except where otherwise stated.

A further aspect of the present invention provides a fragment of afull-length SET polypeptide as described herein, said fragment havinghistone H3 methylase activity.

Preferably such a fragment comprises the SET methylase domain.

A suitable fragment preferably methylates histone H3 outside lysine 9,more preferably at lysine 4 or a lysine residue selected from the groupconsisting of lysine 27, lysine 36, lysine 37, lysine 42, lysine 56,lysine 64, lysine 79, lysine 115, lysine 121, lysine 122 and lysine 125i.e. a core lysine residue of histone H3 located outside the first 26 Nterminal amino acids, for example, the central and C terminal regions,such as lysine 36.

A fragment of a SET polypeptide includes a fragment of a SET1polypeptide, which may be any member of the SET1 family, includinghSET-1P (human KIAA0339) and hSET-2P (human KIAA1076) and a fragment ofa SET2 polypeptide, which may be any member of the SET2 protein family,including hSET2-1, hSET2-2, hSET2-3 and WHSC1.

Another aspect of the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence which encodes a SETpolypeptide. A SET polypeptide may include a fragment of a full-lengthSET polypeptide sequence described herein.

Such a nucleotide sequence may be operably linked to a heterogeneousregulatory element, such as a promoter or enhancer element. A nucleotidesequence encoding a SET polypeptide as described above may be comprisedwithin a vector, in particular an expression vector.

Vectors comprising nucleic acid encoding a SET polypeptide may betransformed into a suitable host cell as described above to provide forexpression of the SET polypeptide. Thus, in a further aspect theinvention provides a process for preparing a SET polypeptide whichincludes cultivating a host cell transformed or transfected with anexpression vector as described above under conditions to provide forexpression by the vector of a coding sequence encoding the SETpolypeptide, and recovering the expressed polypeptide. Polypeptides mayalso be expressed in in vitro systems, such as reticulocyte lysate.

Following production of a SET polypeptide, it may be tested for histoneH3 methytransferase activity, e.g. by determination of methylation oflysine 4 or a core lysine residue of histone H3 selected from the groupconsisting of lysine 27, lysine 36, lysine 37, lysine 42, lysine 56,lysine 64, lysine 79, lysine 115, lysine 121, lysine 122 and lysine 125,preferably lysine 36 as described herein on incubation of thepolypeptide with a histone H3 polypeptide.

Thus the present invention also provides a method of producing a SETpolypeptide comprising;

-   -   expressing a SET polypeptide from encoding nucleic acid, and;    -   determining the methyltransferase activity of the expressed SET        polypeptide.

The methyltransferase activity may include the methylation of a histoneH3 residue other than lysine 9, for example lysine 4 or a lysine residueselected from the group consisting of lysine 27, lysine 36, lysine 37,lysine 42, lysine 56, lysine 64, lysine 79, lysine 115, lysine 121,lysine 122 and lysine 125 i.e. a core lysine residue of histone H3located outside the first 26 N terminal amino acids, for example lysine36. Methyltransferase activity may also include the di- ortri-methylation of such a residue.

It is not necessary to use the entire proteins for assays of theinvention which test for binding between two molecules. Fragments may begenerated and used in any suitable way known to those of skill in theart. Suitable ways of generating fragments include, but are not limitedto, recombinant expression of a fragment from encoding DNA. Suchfragments may be generated by taking encoding DNA, identifying suitablerestriction enzyme recognition sites either side of the portion to beexpressed, and cutting out said portion from the DNA. The portion maythen be operably linked to a suitable promoter in a standardcommercially available expression system. Another recombinant approachis to amplify the relevant portion of the DNA with suitable PCR primers.Small fragments (e.g. up to about 20 or 30 amino acids) may also begenerated using peptide synthesis methods which are well known in theart.

The skilled person can use the techniques described herein and otherswell known in the art to produce large amounts of peptides, for instanceby expression from encoding nucleic acid.

Methods of obtaining agents able to modulate the histone H3 methylationactivity of SET polypeptides include methods wherein a suitableend-point is used to assess interaction in the presence and absence of atest substance.

For methylation assays, a histone H3 or SET polypeptide which is afull-length protein, truncated portion, or a portion of fused to anotherprotein (e.g. GST), or a suitable variant or derivative of any of these,may be used.

Peptide methylation assays may use peptides that comprise the region ofhistone H3 which is methylated. For a SET1 polypeptide, this may includethe N-terminal region of histone H3 comprising lysine 4.

For a SET2 polypeptide, this may include a lysine residue selected fromthe group consisting of lysine 27, lysine 36, lysine 37, lysine 42,lysine 56, lysine 64, lysine 79, lysine 115, lysine 121, lysine 122 andlysine 125, i.e. a core lysine residue of histone H3 located outside thefirst 26 N terminal amino acids, for example, the central and C terminalregions. Preferably, for a SET2 polypeptide, this may include lysine 36.

The methylation of histone H3 may be assayed by any of a variety ofprocedures such as discussed below and may be adapted to high throughputscreening approaches. Of particular interest is the methylation of thelysine 4 residue of histone H3 by hSET-1P (human KIAA0339) and/orhSET-2P (human KIAA1076) and methylation in the central and/or Cterminal portions (i.e. the core) of histone H3 by hSET2(AJ238403)and/or WHSC1 (XP_(—)003388).

A method of the present invention may comprise identifying and/orobtaining a test compound which modulates the histone H3 methylaseactivity of a SET polypeptide.

Following identification of a test compound which is an agent ormodulator as described herein, it may be investigated further. Forexample, a compound, substance or molecule which tests positive forability to modulate methylation of the appropriate residue of histone H3and/or the methylase activity of a SET polypeptide may be isolated,purified and/or manufactured. A method of the present invention may thuscomprise isolating and/or purifying and/or manufacturing or synthesisingthe test compound.

Furthermore, the test compound may be used in preparation, i.e.manufacture or formulation, of a composition such as a medicament,pharmaceutical composition or drug. This may be administered toindividuals. A method of the present invention may thus compriseformulating said compound with a pharmaceutically acceptable excipientas described herein.

In various aspects, the present invention thus provides a modulatoridentified and/or obtained by a screening method of the invention, e.g.a substance which inhibits or reduces, increases or potentiates thehistone H3 methylase activity of a SET polypeptide.

Following identification of a modulator, the substance may be purifiedand/or investigated further and/or manufactured. A modulator may be usedto obtain peptidyl or non-peptidyl mimetics, e.g. by methods well knownto those skilled in the art and discussed herein. It may be used in atherapeutic context as discussed below.

Agents according to the present invention useful in modulating themethylation of histone H3 and therefore one or more of its functionswithin the chromatin, may modulate the methylase activity of the SETpolypeptide. Such agents may specifically inhibit the ability of the SETpolypeptide to methylate the appropriate residue of histone H3 orprovide the appropriate level of methylation. Assays and screens forsuch agents are provided in accordance with the present invention, alongwith the agents themselves and their use in modulating the methylationand thereby the function of histone H3.

An agent able to inhibit methylation of histone H3 by a SET polypeptidemay include a substance able to affect the catalytic properties of theenzymatically active site of the methylase. An inhibitor of methylationmay interact with the SET polypeptide within the SET methylase domain.Residues within this domain are involved in interaction with histone H3and catalysis of the methylation. Residues outside of the domain mayalso be involved in interacting with histone H3 and agents whichinterfere with such interaction may also affect the methylation asdiscussed elsewhere herein.

Agents useful in accordance with the present invention may be identifiedby screening techniques which involve determining whether an agent undertest inhibits or disrupts the methylation by a SET polypeptide of thehistone H3 polypeptide.

One class of putative modulator compounds can be derived from the SETpolypeptide sequence and/or a ligand with which it interacts, such ashistone H3. Peptide fragments of these polypeptides or alleles, mutantsor derivatives of such fragments are described herein. Nucleic acidencoding such peptides, vectors and host cells containing such nucleicacid, and methods of expressing nucleic acid encoding such peptides arefurther aspects of the present invention.

Where the histone H3 polypeptide is a fragment of the full lengthprotein, suitable fragments are those which contain the appropriateresidue which is methylated by a SET polypeptide in the full-lengthpolypeptide, and are themselves capable of being methylated by a SETpolypeptide. Smaller fragments, and analogues and variants of thesefragment may similarly be employed, e.g. as identified using techniquessuch as deletion analysis or alanine scanning.

Fragments methylated by a SET1 polypeptide include fragments comprisingthe N terminal of histone H3, in particular lysine 4. Fragmentsmethylated by a SET2 polypeptide include fragments comprising a lysineresidue selected from the group consisting of lysine 27, lysine 36,lysine 37, lysine 42, lysine 56, lysine 64, lysine 79, lysine 115,lysine 121, lysine 122 and lysine 125 i.e. a core lysine residue ofhistone H3 located outside the first 26 N terminal amino acids, inparticular lysine 36.

Thus, the present invention provides a SET polypeptide, which ispreferably a peptide fragment, which is able to inhibit methylation ofhistone H3.

Such peptide fragments may be obtained by means of deletion analysisand/or alanine scanning of the relevant protein—making an appropriatemutation in sequence, bringing together a SET polypeptide and a histoneH3 polypeptide in the presence of the mutated fragment, and determiningmethylation of the histone H3 polypeptide. In preferred embodiments, thepeptide is short, as discussed below, and may be a minimal portion whichis able to interact with the relevant counterpart protein and inhibitmethylation.

A “fragment” of a polypeptide generally means a stretch of amino acidresidues of at least about five contiguous amino acids, often at leastabout seven contiguous amino acids, typically at least about ninecontiguous amino acids, more preferably at least about 13 contiguousamino acids, and, more preferably, at least about 20 to 30 or morecontiguous amino acids. Fragments of a SET polypeptide may includeantigenic determinants or epitopes useful for raising antibodies to aportion of the amino acid sequence. Alanine scans are commonly used tofind and refine peptide motifs within polypeptides, this involving thesystematic replacement of each residue in turn with the amino acidalanine, followed by an assessment of biological activity.

Where the SET polypeptide is a SET1 polypeptide and the histone H3polypeptide is a fragment, the fragment generally comprises the lysine 4residue.

Where the SET polypeptide is a SET2 polypeptide and the histone H3polypeptide is a fragment, the fragment generally comprises a lysineresidue selected from the group consisting of selected from the groupconsisting of lysine 27, lysine 36, lysine 37, lysine 42, lysine 56,lysine 64, lysine 79, lysine 115, lysine 121, lysine 122 and lysine 125i.e. a core lysine residue of histone H3 located outside the first 26 Nterminal amino acids. More preferably the fragment comprises lysine 36.

Peptides in accordance with the present invention tend to be short, andmay be about 40 amino acids in length or less, preferably about 35 aminoacids in length or less, more preferably about 30 amino acids in length,or less, more preferably about 25 amino acids or less, more preferablyabout 20 amino acids or less, more preferably about 15 amino acids orless, more preferably about 10 amino acids or less, or 9, 8, 7, 6, 5 orless in length. Peptides according to the present invention may be about10–40 amino acids in length, about 5–10, about 10–15, about 10–20, about10–30, about 20–30, or about 30–40 amino acids in length. Peptides whichare histone H3 fragments generally include one or more of the relevantlysine residues.

The present invention also encompasses peptides which are sequencevariants or derivatives of a wild-type SET polypeptide sequence.Peptides which are variants of wild-type SET polypeptide sequence retainthe ability to modulate methylation of histone H3 by a SET polypeptide.

A SET polypeptide or histone H3 polypeptide may be a peptide orpolypeptide which may include an amino acid sequence which differs byone or more amino acid residues from the wild-type amino acid sequence,by one or more of addition, insertion, deletion and substitution of oneor more amino acids. Thus, variants, derivatives, alleles, mutants andhomologues, e.g. from other organisms, are included.

Preferably, the amino acid sequence shares homology with a region of therelevant SET polypeptide or histone H3 polypeptide as referenced herein,preferably at least about 60%, or 70%, or 75%, or 80%, or 85%, 90% or95% homology. Thus, a peptide fragment of a SET polypeptide or histoneH3 polypeptide may include 1, 2, 3, 4, 5, greater than 5, or greaterthan 10 amino acid alterations such as substitutions with respect to thewild-type sequence.

A derivative of a peptide for which the specific sequence is disclosedherein may be in certain embodiments the same length or shorter than thespecific peptide. In other embodiments the peptide sequence or a variantthereof may be included in a larger peptide, as discussed above, whichmay or may not include an additional portion of SET polypeptide orhistone H3 polypeptide. 1, 2, 3, 4 or 5 or more additional amino acids,adjacent to the relevant specific peptide fragment of SET polypeptide orhistone H3 polypeptide, or heterologous thereto may be included at oneend or both ends of the peptide.

Antibodies directed to the site of binding of SET polypeptides formanother class of putative modulators of SET methylase activity.Candidate inhibitor antibodies may be characterised and their bindingregions determined to provide single chain antibodies and fragmentsthereof which are responsible for disrupting the binding.

Antibodies may be obtained using techniques which are standard in theart. Methods of producing antibodies include immunising a mammal (e.g.mouse, rat, rabbit, horse, goat, sheep or monkey) with a SETpolypeptide. Antibodies may be obtained from immunised animals using anyof a variety of techniques known in the art, and screened, preferablyusing binding of antibody to antigen of interest. For instance, Westernblotting techniques or immunoprecipitation may be used (Armitage et al.,1992, Nature 357: 80–82). Isolation of antibodies and/orantibody-producing cells from an animal may be accompanied by a step ofsacrificing the animal.

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a SET polypeptide may be obtained from arecombinantly produced library of expressed immunoglobulin variabledomains, e.g. using lambda bacteriophage or filamentous bacteriophagewhich display functional immunoglobulin binding domains on theirsurfaces; for instance see WO92/01047. The library may be naive, that isconstructed from sequences obtained from an organism which has not beenimmunised with any of the proteins (or fragments), or may be oneconstructed using sequences obtained from an organism which has beenexposed to the antigen of interest.

Antibodies according to the present invention may be modified in anumber of ways. Indeed the term “antibody” should be construed ascovering any binding substance having an immunoglobulin binding domainwith the required specificity. Thus the invention covers antibodyfragments, derivatives, functional equivalents and homologues ofantibodies, including synthetic molecules and molecules whose shapemimics that of an antibody enabling it to bind an antigen or epitope.

A hybridoma producing a monoclonal antibody according to the presentinvention may be subject to genetic mutation or other changes. It willfurther be understood by those skilled in the art that a monoclonalantibody can be subjected to the techniques of recombinant DNAtechnology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP184187A, GB 2188638A orEP-A-0239400. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023.

Hybridomas capable of producing antibody with desired bindingcharacteristics are within the scope of the present invention, as arehost cells, eukaryotic or prokaryotic, containing nucleic acid encodingantibodies (including antibody fragments) and capable of theirexpression. The invention also provides methods of production of theantibodies including growing a cell capable of producing the antibodyunder conditions in which the antibody is produced, and preferablysecreted.

The reactivities of antibodies on a sample may be determined by anyappropriate means. Tagging with individual reporter molecules is onepossibility. The reporter molecules may directly or indirectly generatedetectable, and preferably measurable, signals. The linkage of reportermolecules may be directly or indirectly, covalently, e.g. via a peptidebond or non-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule. The mode of determining binding is not a feature of thepresent invention and those skilled in the art are able to choose asuitable mode according to their preference and general knowledge.

Antibodies may also be used in purifying and/or isolating SETpolypeptide, for instance following production of the polypeptide byexpression from encoding nucleic acid. Antibodies may be useful in atherapeutic context (which may include prophylaxis) to disrupt bindingof a SET polypeptide to histone H3 and inhibit the methylation of thetarget lysine residue. Antibodies can for instance be micro-injectedinto cells, e.g. at a tumour site, subject to radio- and/or chemotherapy(as discussed already above). Antibodies may be employed in accordancewith the present invention for other therapeutic and non-therapeuticpurposes which are discussed elsewhere herein.

As noted, the agent may be peptidyl, e.g. a peptide which includes asequence as recited above, or may be a functional analogue of such apeptide.

As used herein, the expression “functional analogue” relates to peptidevariants or organic compounds having the same functional activity as thepeptide in question, which may interfere with the methylation of thelysine 4 residue of histone H3 by a SET polypeptide. Examples of suchanalogues include chemical compounds which are modelled to resemble thethree dimensional structure of the SET methylase domain in the contactarea, and in particular the arrangement of the key amino acid residues.

In a further aspect, the present invention provides the use of a SETpolypeptide, in particular a peptide fragment, which is capable ofmethylating the lysine 4 residue of histone H3, in a method of designinga peptide or non-peptidyl mimetic, which mimetic is able to interactwith the SET polypeptide active site and modulate the methylation of thelysine 4 residue by the SET polypeptide.

Accordingly, the present invention provides a method of designing amimetic, for example of a histone H3 amino terminal fragment, which hasthe biological activity of modulating the methylation of the lysine 4residue by the SET polypeptide, said method comprising:

-   -   (i) analysing a substance to determine the amino acid residues        essential and important for the biological activity to define a        pharmacophore; and,    -   (ii) modelling the pharmacophore to design and/or screen        candidate mimetics which modulate the methylation as described.

Suitable modelling techniques are known in the art. This includes thestudy of the bonding between SET and histone H3 and the design ofcompounds which contain corresponding functional groups arranged in sucha manner that they could reproduce that bonding.

The designing of mimetics to a known pharmaceutically active compound isa known approach to the development of pharmaceuticals based on a “lead”compound. This might be desirable where the active compound is difficultor expensive to synthesise or where it is unsuitable for a particularmethod of administration, for instance SET polypeptides may not be wellsuited as active agents for oral compositions as they tend to be quicklydegraded by proteases in the alimentary canal.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. Firstly, the particular partsof the compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g. by substituting each residue in turn. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modelledaccording to its physical properties, e.g. stereochemistry, bonding,size and/or charge, using data from a range of sources, e.g.spectroscopic techniques, X-ray diffraction data and NMR. Computationalanalysis, similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

In a variant of the above approach, the three-dimensional structure of aligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this the design of themimetic.

A compound found to have the ability to affect SET methylase activityhas therapeutic and other potential in a number of contexts, asdiscussed. For therapeutic treatment such a compound may be used incombination with any other active substance, e.g. for anti-tumourtherapy another anti-tumour compound or therapy, such as radiotherapy orchemotherapy.

An agent identified using one or more primary screens (e.g. in acell-free system) as having ability to modulate the methylase activityof a SET polypeptide may be assessed further using one or more secondaryscreens. A secondary screen may involve testing for a biologicalfunction of histone H3 methylated at the lysine 4 position or a lysineresidue selected from the group consisting of lysine 27, lysine 36,lysine 37, lysine 42, lysine 56, lysine 64, lysine 79, lysine 115,lysine 121, lysine 122 and lysine 125 i.e. a core lysine residue ofhistone H3 located outside the first 26 N terminal amino acids,preferably lysine 36. Suitable biological functions include thetranscriptional activation of a chromatin region or domain whichcomprises or is associated with the histone H3 polypeptide and/orbinding to a regulatory factor such as the NuRD repressor.

Generally, a modulator according to the present invention is provided inan isolated and/or purified form, i.e. substantially pure. This mayinclude being in a composition where it represents at least about 90%active ingredient, more preferably at least about 95%, more preferablyat least about 98%. Such a composition may, however, include inertcarrier materials or other pharmaceutically and physiologicallyacceptable excipients. As noted below, a composition according to thepresent invention may include in addition to an modulator compound asdisclosed, one or more other molecules of therapeutic use, such as ananti-tumour agent.

The invention further provides a method of treatment which includesadministering to a patient an agent as described herein which interfereswith or inhibits the methylation of the target residue of histone H3(for example lys 4 or a core lys residue) by a SET polypeptide.Exemplary purposes of such treatment are discussed elsewhere herein.

The invention further provides various therapeutic methods and uses ofone or more compounds or substances selected from (i) SET polypeptidewhich is able to bind to histone H3; (ii) a modulator identified by ascreening method of the present invention; (iii) a mimetic of any of theabove substances which can bind to histone H3 or a SET polypeptide.

The therapeutic/prophylactic purpose of such a method or use may be themodulation, e.g. disruption or interference, of the methylation ofhistone H3 at residues other than lysine 9. Target residues includelysine 4 for SET1. For a SET2 polypeptide, this may include a lysineresidue selected from the group consisting of lysine 27, lysine 36,lysine 37, lysine 42, lysine 56, lysine 64, lysine 79, lysine 115,lysine 121, lysine 122 and lysine 125 i.e. a core lysine residue ofhistone H3 located outside the first 26 N terminal amino acids, forexample, within the central and C terminal regions, preferably lysine36.

The therapeutic/prophylactic purpose may be:

-   (i) Cancer treatment, which may for example be in combination with    chemotherapy and/or radiotherapy.-   (ii) Cancer prophylaxis,-   (iii) Treatment of other proliferative disorders described herein    e.g. psoriasis, cataracts, multiple myeloma.

In various further aspects, the present invention thus provides apharmaceutical composition, medicament, drug or other composition forsuch a purpose, the composition comprising one or more such compounds orsubstances which modulate the activity of a SET polypeptide as describedherein and/or the methylation of histone H3 at a lysine residue otherthan Lys9, the use of such a substance in a method of medical treatment,a method comprising administration of such a substance to a patient,e.g. for treatment (which may include preventative treatment) of amedical condition, e.g. a condition associated with a defect or disorderin transcriptional control, DNA replication, or cell cycle control, e.g.for treatment of a disorder of cellular proliferation such asrestenosis, psoriasis, cataracts, multiple myeloma and cancer,preferably all types of solid cancers and malignant lymphomas andespecially leukaemia, skin cancer, bladder cancer, breast cancer, uteruscancer, ovary cancer, prostate cancer, lung cancer, colon cancer,pancreas cancer, renal cancer, stomach cancer and cerebral cancer, useof such a substance in the manufacture of a composition, medicament ordrug for administration for such a purpose, e.g. for treatment of aproliferative disorder, and a method of making a pharmaceuticalcomposition comprising admixing such a substance with a pharmaceuticallyacceptable excipient, vehicle or carrier, and optionally otheringredients.

The substances may be used as sole active agents or in combination withone another or with any other active substance, e.g. for anti-tumourtherapy another anti-tumour compound or therapy, such as radiotherapy orchemotherapy.

Whatever the substance used in a method of medical treatment of thepresent invention, administration is preferably in a “prophylacticallyeffective amount” or a “therapeutically effective amount” (as the casemay be, although prophylaxis may be considered therapy), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated. Prescription oftreatment, e.g. decisions on dosage etc, is within the responsibility ofgeneral practitioners and other medical doctors.

A substance or composition may be administered alone or in combinationwith other treatments, either simultaneously or sequentially dependentupon the condition to be treated, e.g. cancer.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may include, in additionto active ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabiliser or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the rouse of administration, which maybe oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Liposomes, particularly cationic liposomes, may be used in carrierformulations.

Examples of techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

The substance or composition may be administered in a localised mannerto a tumour site or other desired site or may be delivered in a mannerin which it targets tumour or other cells.

Targeting therapies may be used to deliver the active substance morespecifically to certain types of cell, by the use of targeting systemssuch as antibody or cell specific ligands. Targeting may be desirablefor a variety of reasons, for example if the agent is unacceptablytoxic, or if it would otherwise require too high a dosage, or if itwould not otherwise be able to enter the target cells.

Instead of administering such substances directly, they may be producedin the target cells by expression from an encoding nucleic acidintroduced into the cells, e.g. from a viral vector. The vector may betargeted to the specific cells to be treated, or it may containregulatory elements which are switched on more or less selectively bythe target cells.

Nucleic acid encoding the substance e.g. a peptide able to modulate,e.g. interfere with, the methylation of the lysine 4 or core lysineresidue of histone H3 by a SET polypeptide, may thus be used in methodsof gene therapy, for instance in treatment of individuals, e.g. with theaim of preventing or curing (wholly or partially) a disorder.

Vectors such as viral vectors have been used in the prior art tointroduce nucleic acid into a wide variety of different target cells.Typically the vectors are exposed to the target cells so thattransfection can take place in a sufficient proportion of the cells toprovide a useful therapeutic or prophylactic effect from the expressionof the desired peptide. The transfected nucleic acid may be permanentlyincorporated into the genome of each of the targeted cells, providinglong lasting effect, or alternatively the treatment may have to berepeated periodically.

A variety of vectors, both viral vectors and plasmid vectors, are knownin the art, see U.S. Pat. No. 5,252,479 and WO 93/07282. In particular,a number of viruses have been used as gene transfer vectors, includingpapovaviruses, such as SV40, vaccinia virus, herpesviruses, includingHSV and EBV, and retroviruses. Many gene therapy protocols in the priorart have used disabled murine retroviruses.

As an alternative to the use of viral vectors in gene therapy otherknown methods of introducing nucleic acid into cells includes mechanicaltechniques such as microinjection, transfer mediated by liposomes andreceptor-mediated DNA transfer.

Receptor-mediated gene transfer, in which the nucleic acid is linked toa protein ligand via polylysine, with the ligand being specific for areceptor present on the surface of the target cells, is an example of atechnique for specifically targeting nucleic acid to particular cells.

A peptide or other substance having an ability to modulate or interferewith the methylation of the lysine 4 or core lysine residue of histoneH3 by a SET polypeptide, a nucleic acid molecule which encodes a peptidehaving that ability, may be provided in a kit, e.g. sealed in a suitablecontainer which protects its contents from the external environment.Such a kit may include instructions for use.

In still further aspects the present invention provides for thepurification of a SET polypeptide, or for the purification of a histoneH3 polypeptide. The invention also provides for a purified SETpolypeptide protein. The purified polypeptide may be about 10% pure,more preferably about 20% pure, more preferably about 30% pure, morepreferably about 40% pure, more preferably about 50% pure, morepreferably about 60% pure, more preferably about 70% pure, morepreferably about 80% pure, more preferably about 90% pure, morepreferably about 95% pure, or substantially pure.

In another aspect the present invention provides a method of purifying aSET polypeptide, the method including contacting the SET polypeptidewith histone H3 polypeptide.

A mixture of material including SET polypeptide may be contacted againstimmobilised histone H3 polypeptide (e.g. immobilised either covalentlyor non-covalently such as via a specific binding molecule such asstreptavidin or biotin) and molecules which do not bind to the histoneH3 polypeptide are washed off.

Following purification, the SET polypeptide may be used as desired, e.g.in an assay for an agent which modulates its activity, e.g. binding, inraising or obtaining a specific antibody or other binding molecule, orin a therapeutic context.

Other aspects of the present invention relate to the disruption of theinteraction between histone H3 and the NuRD complex by the methylationof histone H3 lysine 4 by a SET1 polypeptide.

A method for identifying and/or obtaining an agent which modulates theinteraction of histone H3 with a NuRD repressor, may include:

-   (a) bringing into contact one or more components of the NuRD    repressor complex and a histone H3 polypeptide in the presence of a    test compound; and,-   (b) determining interaction between the histone H3 polypeptide and    the said one or more components.

The one or more components of the NuRD repressor complex and the histoneH3 polypeptide may be brought together in the presence of a SET1polypeptide. A reduction in the activity of the SET1 polypeptide inmethylating the lys 4 residue of the histone H3 polypeptide may bedetermined by an increase in interaction and/or binding between the NuRDcomplex and the histone H3 polypeptide in the presence relative to theabsence of the test compound.

Components of the NuRD complex may include HDAC1, HDAC2, Mi-2β, Rbap48,Rbap46, MTA1, MTA2 and MBD3 (Zhang et al (1998) Cell 95(2)279–289).Histone H3 polypeptides are described above.

Interaction and/or binding may be determined by any convenient techniqueas described above. A agent identified and/or obtained using such amethod may be useful in the modulation of transcriptional activation.

Other aspects of the present invention relate to the tri-methylation ofhistone H3 lysine 4 by a SET1 polypeptide and the effect of lys 4tri-methylation on transcriptional activation.

A method of identifying transcriptionally active chromatin may comprise;

-   -   determining the presence or absence of a trimethylated Lys 4        residue in a histone H3 polypeptide of said chromatin.

The presence of a trimethylated Lys4 residue in a histone H3 polypeptideof said chromatin is indicative that said chromatin is transcriptionallyactive.

The presence or absence of a trimethylated Lys 4 may be determined byany convenient means. For example, said chromatin region may becontacted with an antibody which binds specifically to a histone H3polypeptide which is trimethylated at Lys 4.

Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the figure described below.

FIG. 1 shows sequence alignments within the SET domains and flankingsequences of yeast proteins containing SET domains, human homlogues ofSET1 and the human histone H3 lysine 9 methylase Suv39h1. hSet1.1, humanKiAA0339; hSet1.2, human KIAA1076; sp Set1, S. pombe SPCC306.04c; scSet1, S. cerevisiae Set1p; sc Set2, S. cerevisiae Set2p; Suv39H1, humanSuvar 39H1; sc Set3, S. cerevisiae YJL105W; sc Set4, S. cerevisiaeYKRO29C; sc Set5, S. cerevisiae YHR207C; sc Set6, S. cerevisiae YPL165C.Alignments were calculated as follows: Clustal W1.8 (DNA-Protein)-Globalprogressive (BCM), BOXADE 3.21, Consensus line: No consensus, Fractionof sequences (that must agree for shading): 0.5.

FIG. 2 shows sequence alignments within the SET domains and flankingsequences of SET1 and SET2 polypeptides. hSet1.1, human KiAA0339;hSet1.2, human KIAA1076; sc Sett, S. cerevisiae Set1p; spSet2, S. pombeSPAC29B12; sc Set2, S. cerevisiae Set2p; hSet2.1, human KlAA1732;hSet2.2, human HSPCO69; hSet2.3, human AJ238403; WHSC17 human WHSC1.

FIG. 3 shows the mapping of the methylation site of the S. cerevisiaeSET1 in the histone H3 amino tail.

FIG. 4 shows a relationship tree between proteins which contain SETdomains. The tree was compiled using the following multiple alignmentparameters; program—Clusta1W (v1.4), similarity matrix—blosum, Open Gappenalty—10.0, Extend Gap penalty—0.1, Delay Divergent—40%, Gapdistance—8.

FIG. 5 shows a schematic diagram of various methylated and unmethylatedhistone H3 peptides used to purify a specific set of binding proteins.The C-terminus of each of the peptides was followed by GGC.

FIG. 6 shows the deacetylase activity and released tritiated acetate, asmeasured by scintillation counting (c pm=counts per minute) fromproteins bound to the unmethylated and methylated histone H3 peptide inpull down assays.

FIG. 7 shows a list of genes activated by SET1. To identify targets ofSET1 mediated expression, transcription profiles of set1Δ yeast weredetermined. Potential targets were collated from genes down regulated inthe mutant and assigned relative merit (P-value) using a gene-specificerror model. Secondary effects were eliminated by disregarding genesalso down regulated in general stress or in a sir2Δ profile (sir2Δ andsir1Δ yeast both exhibit downstream effects secondary to derepression ofthe silent mating loci).

Table 1 shows % identity within the SET Domain among the SET1 and SET2Families. The table was compiled using the following parameters;program—MacVector 6.5, matrix—BLOSUM 30, Clustal alignment—pairwisealignment, Open Gap penalty—1.0, Extend Gap penalty—0.1.

Table 2 shows a summary of the K4 methylation state at the promoter ofconstitutive genes, inositol regulated genes and methionine regulatedgenes. “−” means no methylation detected. “Di” means di-methylationdetected at the promoter and “tri” means tri-methylation at thepromoter. The growth conditions for activation and repression of thegenes are described below.

EXPERIMENTAL

Materials and Methods

Purification of Yeast SET Protein

Yeast Set1p and Set6p were tagged at the N-terminus with a Protein Aepitope from S. aureus. These proteins were expressed in the wild typestrain RS453 (mat a, ade2, ura3, leu2, his3, trp1) and in the C 13ABYS-86 strain (Matα ura3 leu2-3 his3-112 pra1-1 prb1-1 prc1-1 cps1-3).The tagged proteins with the expected size (PtA-Set1p, 160 Kda;PtA-Set6p, 55 Kda) were detected in total extract by western blot withAnti Protein A antibody (PAP, rabbit, DAKO-Z0113) 1:3000 dilution.PtA-Set1p and PtA-Set6p were purified by affinity to an IgG Sepharosecolumn as described (Siniossoglou, S. et al (1996) Cell 84, 265–275).For each set of assays, 5 g of spheroplasts were homogenised in 50 ml oflysis buffer. The soluble supernatant (S) was passed through a columncontaining 200 μl of IgG beads.

Avoiding the elution step, the beads were washed with 25 ml ofequilibration buffer. Then, 60 μl of beads were transferred to 3eppendorf tubes, placed on ice, for the enzymatic reaction. Theremaining 20 μl beads were re-suspended in 50 μl Laemli buffer, vortexand boiled for 3 min. Samples of “soluble supernatant, S” (30 μl,equivalent to 0.06% of the total soluble supernatant) and “elutedprotein, E” (25 μl, equivalent to 5% of the total purified protein) wereanalysed by western blot with Anti Protein A antibody (PAP, rabbit,DAKO- Z0113) 1:3000 dilution.

Equilibration Buffer: 20 mM Tris-HCl pH 8.5; 0.05 mM DTT.

Methyltransferase Assay

The enzymatic assays were performed on a mixture of soluble histonesfrom calf thymus (H2A, H2B, H3 and H4), Sigma; recombinant xenopus H3 orH3 deleted for the first 26 amino acids (Δtail H3).

For each reaction, 60 μg of calf histones or 20 μg of recombinant H3were used. The following mixtures were prepared:

-   1) 60 μl of PtA-Set p beads (either Set1p, Set2p or Set6p)+30 μl of    methylation buffer+2 μl of ¹⁴C-SAM (NEC-363 adenosyl-L-methionine,    S-(methyl-¹⁴C).-   2) 60 μl of PtA-Set p beads (either Set1p, Set2p or Set6p)+30 μl of    methylation buffer+3 μl of soluble histones (60 μg)+2 μl of 14C-SAM    (NEC-363 adenosyl-L-methionine, S-(methyl-¹⁴C).-   3) 60 μl of PtA-Set p beads (either Set1p, Set2p or Set6p)+30 μl of    methylation buffer+4 μl of recombinant xH3 (20 μg)+2 μl of 14C-SAM    (NEC-363 adenosyl-Lmethionine, S-(methyl-14C).-   4) 60 μl of IgG sepharose beads (previously equilibrated in    methylation buffer)+30 μl of methylation buffer+3 μl of soluble    histones (60 μg)+2 μl of 14C-SAM (NEC-363 adenosyl-L-methionine,    S-(methyl-14C).-   5) 60 μl of IgG sepharose beads (previously equilibrated in    methylation buffer)+30 μl of methylation buffer+4 μl of recombinant    xH3 (20 μg)+2 μl of 14C-SAM (NEC-363 adenosyl-L-methionine,    S-(methyl-14C).

Methylation Buffer: 50 mM Tris-HCl pH 8.5; 20 mM K Cl, 10 mM MgCl2, 10mM -mercaptoethanol, 0.05 mM DTT, 250 mM sucrose, 0.2%dodecyl-β-D-maltoside.

When assayed, the recombinant xenopus H3 deleted for the 26 first aminoacids (xgH3) was used at 10 μg/reaction.

The microfuge tubes were incubated at 30° C. during 2 hours withshaking. After that, 15 μl of 4× (protein sample buffer) were added toeach reaction (the total liquid vol. of each reaction should be around45 μl). The samples were boiled for 3 minutes. The histones wereseparated in a 1.5 mm, 20% acrylamide gel, run at 200 V during 1 hourand 15 minutes and transferred to nitro-cellulose membranes by standardprocedures. The nitro-cellulose membrane was exposed to Kodak Biomax MSfilm with Biomax Transcreen-LE intensifying screen at −80 C during 36–48h.

Mapping of Site of Methylation within Histone H3

Histone H3 was methylated by SET1 as described herein with a ¹⁴⁻C-methylgroup. The methylated residue was then identified using N terminalsequence analysis as described in Rein D. and Speicher D. CurrentProtocols in Protein Science (1997) 11.10.01–11.10.38.

Briefly, A stained protein band was excised from a PVDF membrane,sonicated in a both sonicator and sequenced using a Hewlett-PackardModel G 100SA sequencing system. Sequencing was carried out according tothe manufacturer's instructions and PTH (phenylthiohydantoin) aminoacids from each sequencer cycle were separated by in-line HPLC toprovide sequence data.

Expression Profiling

The set1Δ mutant and its parent strain UCC1001 were grown in parallel inYPD media at 30° C. to an OD₆₀₀ of 1.0. Poly (A) RNA was isolated andreverse transcribed incorporating amino-allyl dUTP. The resulting DNAprobe was labeled with reactive Cy5 (mutant) or Cy3 (parent strain) dyeand hybridized to a spotted cDNA microarray containing the yeast openreading frames as described in Carroll et al (2001) PNAS 98 12578–12583and DeRisi et al (1997) Science 278 680–686. Microarrays were analyzedusing a GenePix4000A scanner and GENEPIX 3.0 software. Candidate SETIregulated genes were identified from duplicate experiments using a genespecific error model described previously (Hughes et al Cell (2000) 102109–126).

Northern Blots and ChIPs

Northern blots and ChiPs were done as described in Kent et al Genes Dev(2001) 15 619–626, except that for ChIP 5μl of Tri-meK4 H3 antibody and2 μl of Di-me K4 H3 antibody were used and protein A-sepharose beadswere pre-incubated with 1.5 μg of sonicated salmon sperm DNA for 30minutes. For the ChIPs experiments, the cell were grown o/n in minimalmedium lacking inositol (medium −ino), then diluted to OD600: 0.15 inminimal medium −inositol or the same one supplemented with 100 mg/Linositol (medium +ino) and grown to OD600: 1.2 and then processed forchromatin preparation.

Samples from medium −methionine and +methionine were prepared in thesame way except that the medium was supplemented.

Affinity Purification from Hela Nuclear Extract

Histone peptides which were trimethylated at different lysine residues,were made using standard techniques. The C-terminus of the peptidescontained a 2 glycine spacer followed by a cysteine. The peptides wereimmobilised onto Sulfolink Coupling gel (Pierce) via the C-terminalcysteine, at a concentration of 1 mg/ml.

Hela nuclear extract (Computer Cell Culture Centre, Belgium) was dilutedin IPH buffer (50 mM Tris pH8, 150 mM NaCI, 5 mM EDT A. 0.5% NP-40 v/v)to a final protein concentration of 4 mg/ml and pre-cleared.

Affinity purifications were done with 5 μl of sepharose-linked peptideand 150 μl of diluted Hela nuclear extract. Competitor peptide was addedto a final concentration of 150 μg/ml, where used. The purificationswere incubated for 90 minutes on a wheel at 4° C. and washed 3 times inIPH before resolution on an 8% SDS-PAGE gel. For column purification ofcomplex, 2 ml of diluted Hela nuclear extract was poured twice over amicro-column of 100 μl of sepharose-linked unmethylated H3 peptide. Thecolumn was washed with 4 column volumes of IPH and bound proteins wereeluted in 250 mM NaCl IPH. Eluted fractions containing the complex weremade up to 150 μl in IPH and rebound to 5 μl of H3 peptide sepharose, asabove. Bound proteins were visualised by silver staining as describedpreviously (Morrissey (1981) 117(2) 307–310).

Mass Spectrometry Identification of Proteins

Proteins that rebound un-methylated peptide after column purificationwere excised from an 8% coomassie stained gel with a protein-free razorblade. Proteins were digested in-gel with trypsin. After overnightincubation at 30° C., a 0.5 μl aliquot from the digest was spotted onthe MALDI target for fingerprinting. The remaining digest was purifiedusing C18 Zip-Tips prior ESI-MS. Peptide Mass Fingerprints were takenwith a MALDI instrument from Micromas fitted with Delayed Extraction,using Alpha-cyano-cinnamic acid dissolved in 50% Acetonitrile: 0.1% TFAas matrix. CID fragmentation spectra from peptides were taken with anIon-trap instrument (LCQ-Decca) from ThermoQuest. Protein databases weresearched with the mass spectrometry data using the programs Mascot,http://matrixscience.com (Mass fingerprinting and fragmentation spectra)and Profound, http://129.85.19.192 (mass fingerprinting data).

Deacetylase Assays

Histone peptide affinity purifications from Hela nuclear extract werere-suspended in 100 μl of fresh IPH buffer containing 150,000 cpm of[³H]-labelled acetylated H4 peptide. Reactions were incubated at 37° C.for 120 minutes, with regular mixing. Reactions were stopped by theaddition of 65 μl of acid mix (1 M HCl, 0.16 M acetic acid). 700 μl ofethylacetate was added and tubes were vortexed vigorously for 20seconds. Phases were separated by centrifugation at 13500 rpm for 1 min.500 μl of the upper phase (ethylacetate) was removed and mixed with 1 mlof scintillant (Optiphase™, Wallac). Release of (³H]-acetate from the H4peptide was measured in counts per minute (cpm) using a scintillationcounter (Beckmann LS6000SC).

Western Blots, Antibodies and Immunoprecipitations

Bound proteins were resolved on 8% SDS-PAGE gel, blotted tonitrocellulose and blocked overnight (4% non-fat milk/0.5% Tween 20v/v). Blots were probed with the following antibodies for 1 hour at roomtemperature: anti-Rbap48 (Genetex, 11g10, 1 μg/ml), anti-DNMT1 (NEB-231,1 in 1000), anti-Sin3 (Santa Cruz 994, 1 μg/ml) and anti-p60 (1 in1000). Blots were washed in blocking buffer, incubated with HRP linkedsecondary antibodies (Abcam) and visualised with ECL (Amersham). NuRDimmunoprecipitations were done by incubating 500 μI of diluted Helanuclear extract with anti-MTA2 antibody (Santa Cruz 9447: 1 μg/ml) and15 μl of protein A/G bead mix (Amersham) for 5–12 hours at 4° C.

Precipitations were washed 3 times with IPH before resolution on a 20%SDS-PAGE gel. After blotting to nitrocellulose and blocking (5% BSA/0.5%Tween 20) for 1 hour at room temperature, the blots were probedovernight with anti-MBD3 (Santa Cruz 9402, 1 μg/ml), anti-H3 (500 ng/ml)or anti-H4 (Abcam ab7311, 500 ng/ml).

Blots were washed briefly with BSA blocking buffer, incubated with HRPlinked secondary antibody and visualised as above.

Results

S. cerevisiae SET1 and SET2 are Histone

Methyltransferases (HMTs)

PtA tagged SET1p, SET2p and SET6p were purified from S. cerevisiae byIgG sepharose chromatography. Western blotting revealed a strong band ineach of the eluted protein fractions corresponding to full-lengthPtA-SET1p, PtA-SET2p and PtA-SET3p, respectively.

Approximately 10 μg of purified PtA-SET1p, PtA-SET2p or PtA-SET6p wereassayed for methyltransferase activity on 60 μg of a mixture of calfthymus soluble histones (H2A, H2B, H3 and H4) or 20 μg of recombinantxenopus histone H3 in the presence of (¹⁴C-Me) S-adenosyl methionine.Autoradiograms were developed after 48 hours exposure.

No methylation was observed for PtA-SET1p, PtA-SET2p or PtA-SET6p in theabsence of calf or recombinant histones.

PtA-SET1p and PtA-SET2p were observed to methylate recombinant Xenopushistone H3. When the preparation of histones H2A, H2B; H3 and H4 wasused, PtA-SET1p and PtA-SET2p were observed to specifically methylatehistone H3.

No methylation activity was observed for PtA-SET6p in the presence ofhistones H2A, H2B, H3 and H4 or the presence of recombinant Xenopushistone H3.

The Methyltransferase Activity of SET1 is Specific for Lysine 4.

A truncated recombinant Xenopus H3 histone containing lacking the first26 N terminal residues was compared with the full length Xenopus H3histone as a substrate for PtA-SET1p and PtA-SET2p.

For each assay, 20 μg of recombinant Xenopus H3 histone and 10 μg ofPtA-SET1p were incubated in the presence of (¹⁴C-Me) S-adenosylmethionine. Autoradiograms showing the presence of ¹⁴C label weredeveloped after 48 hours. The presence of substrates on the gel wasconfirmed by Ponceau staining.

No methylation was observed in the absence of a histone H3 substrate.

Both PtA-SET1p and PtA-SET2p were observed to methylate full lengthXenopus histone H3.

No methylation by PtA-SET1p of the Xenopus histone H3 with the Nterminal deletion was observed but PtA-SET2p was observed to methylatethis truncated protein.

These results demonstrate that PtA-SET1p methylates histone H3 withinthe N terminal 26 amino acid residues of histone H3 whereas PtA-SET2pmethylates outside this amino tail region, within the core of thehistone H3 polypeptide.

Mapping of the Methylation Site within the H3 Amino Tail

Xenopus H3 histone labelled with a ¹⁴C-methyl group by PtA-SET1p asdescribed above, was sequenced by sequential Edman degradation andfractions corresponding to each amino acid cycle were collected andcounted by scintillation counting. The results are shown in FIG. 3. TheN terminal sequence of Xenopus histone H3 is shown underneathcorresponding fraction.

These results show that the lysine 4 residue of histone H3 isspecifically methylated by PtA-SET1p. No methylation of lysine 9 isobserved.

SET polypeptides as described herein are shown to specifically methylatehistone H3 at residues other than lysine 9.

SET1 polypeptides methylate the lysine 4 residue while SET2 polypeptidesmethylate the core of histone H3, outside the amino tail.

The Histone H3 N-terminus Binds a Set of Proteins When Unmethylated atLysine 4

Differently methylated histone peptides were used in pull downs assaysfrom Hela nuclear extract and the bound proteins analysed by silverstain.

The histone peptides used were trimethylated at lysine 4 (K4), lysine 9(K9) or both (K4+K9) and two control peptides of unmethylated H3 and H4tails (FIG. 5). These peptides were immobilised onto sepharose beads viaa C-terminal cysteine residue and used to affinity purify proteins fromHela nuclear extract.

Although some proteins were found to specifically bind to the methylatedpeptides, a number of proteins were also observed to be specificallypurified by the unmethylated H3 peptide.

This set of proteins was also observed to be purified by the K9methylated H3 peptide, but not by a peptide methylated at K4, or K4 andK9, or by an unmethylated H4 peptide. This indicates that these proteinsonly bind to histone H3 N-termini when lysine 4 is unmethylated and thatmethylation of lysine 9 does not disrupt the binding of the complex.

To confirm the specificity of the proteins binding to unmethylated H3peptide, pull downs were done in the presence or absence of competitorpeptide. The set of proteins bound to the unmethylated peptide wasobserved to be competed away by peptides unmethylated at K4, but not bypeptides with a methylated K4.

The Set of Proteins Bound to Unmethylated H3 Tails is the NuRD Complex

The set of proteins, putatively components of a binding complex,identified above were purified from Hela nuclear extract over a columnof unmethylated H3 peptide. The column-purified proteins, once eluted inhigh salt, were rebound to unmethylated peptide in a pull down assay.The rebound proteins were resolved by SDS-PAGE and silver stained. Thispurified source of the putative complex was used for mass spectrometryidentification of the components.

The sub-units of the complex identified in this way include the histonedeacetylases HDAC1/2, the ATPase chromatin-remodelling enzyme Mi-2β, theRB associated proteins Rbap48/46, the metastasis-associated antigensMTA1/2 and the methyl CpG binding domain MBD3. These are all knowncomponents of the NuRD complex (Zhang et al (1998) Cell 95(2) 279–289,Zhang et al (1999) Genes Dev 13(15) 1924–1935, Xue et al (1998) Mol Cell2(6) 851–861).

Several components of the NuRD complex have been identified in otherdeacetylase complexes (Knoepfler, P. S. et al (1999) Cell 99, 447–450).The purified complex with antibodies against other deacetylase complexcomponents. Rbap48 protein was detected, whereas DNMT1 and Sin3 (presentin distinct deacetylase complexes: Robertson, K. D. et al (2000) NatGenet 25(3), 338–42. 22, Zhang, Y et al (1997) Cell 89(3), 357–64), wereabsent from the H3 purified complex. Rbap48 is also present in the CAF-1(chromatin assembly factor 1) complex (Ridgway, P. et al (2000) J CellSci 113{pt 15), 2647–58.). However a western blot with the p6O subunitof CAF-1 did not detect CAF-1 in the H3 purified complex.

To establish if NuRD has deacetylase activity when affinity purified onunmethylated H3 peptide, peptide pull downs were assayed for deacetylaseactivity. Unmethylated H3 peptide was found to associate with asignificant level of histone deacetylase (HDAC) activity (FIG. 6). Incontrast, a peptide methylated at K4 pulled down background levels ofHDAC activity. This indicates that the deacetylase activity of the NuRDcomplex is not compromised when it is associated with the unmethylatedhistone H3 tail.

NuRD Complex Associates with Native Histone H3 Only When Unmethylated atLysine 4

Previous studies have shown that the NuRD complex can beimmunoprecipitated using anti-MTA2 antibodies (Zhang et al (1999)supra). MT A2 antibodies were observed to precipitate MBD3 (a componentof NuRD) as well as histone H3 and H4, as detected by western blot withthe respective antibodies.

The immunoprecipitated histones were found to be efficiently competedaway by the unmethylated peptide, but not by the K4 methylated peptide.These data indicate that the NuRD complex associates with unmethylatednative histone H3 (and interacts indirectly with H4 via H3).

Methylation Status of K4 of Histone H3

The ε amino group of lysine residues may be mono-, di- or tri-methylated(me). Antibodies were raised to specifically recognize either the di-meor tri-me state of K4 H3. An antibody raised against di-me K4 was foundto recognize H3 from a purified source of histones and is effectivelycompeted by a di-me but not a tri-me K4 H3 peptide. An antibody raisedagainst tri-me K4 was found to be specifically competed by the tri-mebut not di-me K4 H3 peptide. Both antibodies were found to selectivelyrecognise histone H3 in a total yeast extract. Neither antibodyrecognised H3 from a yeast strain containing a K4 H3 mutation to alanine(K4A) and neither antibody recognised bacterially produced recombinant,unmethylated H3.

Both Di- and Tri-me K4 antibodies were found to recognise histone H3after it has been methylated by SET1. Deletion of SET1 abolishesrecognition of histone H3 by the Di- and Tri-me K4 antibodies, whereasdeletion of other S. cerevisiae SET genes (SET2, 3, 4, 5 and 6) has noeffect.

To establish if the bi- or Tri-me state is linked to gene activity, weinvestigated whether SETI can act as an activator of transcription andidentified genes positively regulated by SET1.

Transcriptional profiling of yeast deficient in SET1 waS thereforecarried out and 200 genes were identified whose activity is reduced inthe absence of SET1. FIG. 7 shows a list of the top twenty genes whosetranscription is reduced between 1.6 and 2.1 fold in set1Δ strainrelative to WT yeast. Northern blot analysis of one of these genes PPH3,confirms that the mRNA expression of this gene is indeed reduced in theset1Δ strain. This indicates that SET1 positively regulatestranscription.

We next established whether the SET1 activated genes were methylated atK4H3 and identified the methylation state of K4. The PPH3 gene, as wellas two of the other SET1-dependent genes, HAM1 and NUP 170, wereinvestigated. The use of Di-me and Tri-me specific antibodies inchromatin immunoprecipitation (ChiPs) showed that all three genes haveDi- and Tri-me K4 H3 associated with their promoter.

Since the expression profiling was done from yeast grown in rich medium,all three of these genes, and indeed all the SET1-regulated genes inFIG. 7, are constitutively active.

The K4 H3 methylation status of a set of genes whose activity can beregulated was then investigated using genes that can be activated byeither inositol or methionine deprivation. When the INO1 gene isrepressed, K4 H3 was found to be Di-me but not Tri-me. However, when thegene is active, K4 H3 Di-me was still observed but Tri-me also becameapparent. Two other inositol regulated genes INO2 and INO4 also showedthe presence of Tri-me K4 H3 only when active.

Analysis of an independent metabolic pathway, where genes are activelytranscribed in the absence of methionine, showed that the MET16 gene hasTri-me K4 H3 only when the gene is active and Di-me only when repressed.Another methionine regulated gene, METI7, showed no Di-me K4H3, eitherwhen active or repressed but tri-me K4 H3 appeared when the gene becameactive. The K4 methylation patterns we observed at all the genesanalysed here were not restricted to promoter regions but were alsoobserved when probes in the coding region were used.

These results (summarized in Table 2) indicate that the trimethyl stateof K4H3 is unambiguously linked to active transcription. When genes arerepressed, tri-me K4 H3 is not observed. In contrast, Di-me K4 H3 isoften present in repressed genes but is also present on some activegenes. This is consistent with previously reported findings at thechicken beta-globin locus, where Di-me K4 H3 is detected on largeeuchromatic regions encompassing both active and inactive genes (Litt etal Science 293 2453–5 (2001)). Di-me K4 H3 may determine atranscriptionally active state “poised” for stimulation.

These findings demonstrate that SET1 is an activator of transcriptionand uncover a new level of regulation of K4 H3 function, by virtue ofmethylation status.

Methylation of histone H3 by SET polypeptides plays an important role inthe regulation of transcriptional activity and the control of cellularproliferation. Manipulation of the SET polypeptide histonemethyltransferase activity may therefore be used in the treatment ofconditions associated with cellular proliferation, such as cancer.

TABLE 1 scSET1 pSET1 hSET1.1 hSET1.2 scSET2 spSET2 hSET2 WHSC1 Suv39HscSET1 100%  51% 57% 54%   29% 30% 28%   33% 28% spSET1 100% 55% 54%  29% 32% 30%   30% 28% hSET1.1 100%  82% 25.5% 28% 28% 27.5% 25%hSET1.2 100%    27% 30% 30%   31% 24% scSET2  100% 61% 47% 41.5% 31%spSET2 100%  49%   40% 29% hSET2 100%    49% 31% WHSC1  100% 30% Suv39H100% 

TABLE 2

1. A method of identifying transcriptionally active chromatincomprising: contacting chromatin with an antibody that bindsspecifically to a histone H3 polypeptide which is trimethylated at Lys4,and determining the presence or absence of binding of said antibody tosaid chromatin, the presence of binding of said antibody to saidchromatin being indicative that said chromatin is transcriptionallyactive chromatin.